Nowadays, an emerging challenge is not to produce single proteins only, but to implement whole pathways into microorganisms. Such engineered strains provide new opportunities in industrial processes e.g. for the production of valuable building blocks, derivatives of complex secondary metabolites or to improve cellular functions where multiple proteins are involved such as protein folding, secretion and resistance to environmental stress. For this purpose the genetic stability of production strains is of major importance.
Up to now, expressing three or more genes in P. pastoris is mainly achieved by employing the same regulatory elements. However, the repeated use of homologous sequences can result in recombination events and thus in genetic instability [1]. In addition, the transformation rates of microbial cells usually decrease with increasing the size of the expression constructs while technological difficulties and the costs for labor and materials increase with the size. However, most published engineered and synthetic pathways comprise sets of three to five additional genes which need to be coexpressed, while pathways of natural secondary metabolites are usually even longer.
One strategy to reduce the loss of genes by homologous recombination is the use of different promoter and terminator sequences for each individual gene of the pathway. Alternatively, the number of regulatory elements can be reduced by the expression of multiple genes from a single, polycistronic transcript. While this is simple to achieve in prokaryotes, this is more difficult for eukaryotes. However, such a coordinate expression can be achieved by employing self-processing 2A sequences [2]. 2A sequences are short peptides (up to 20 amino acids) originating from viral polyproteins. They are supposed to cause a ribosome “skip” resulting in the cleavage of the polycistronic transcript between the Gly and the Pro at the C-terminus of the 2A sequence [3]. 2A sequences have already been successfully employed for polycistronic expression in various hosts including the yeast S. cerevisiae up to three proteins were produced employing this strategy [4, 5] and also P. pastoris (Komagattaella phaffi) where 2 proteins have been coexpressed from the same vector using 2A technology [6-8]. Four genes have been coexpressed. To avoid nonstoichiometric expression of multiple proteins as known for genes coupled by IRES sequences Szymczak et al expressed 4 CD3 proteins linked by 2A sequences and showed stoichiometric production of two fluorescent proteins in multiple cell types [9].
Heterologous gene expression of up to three gene copies in a mushroom, Flammulina velutipes using polycistronic vectors was described [16] and the use of 2A peptides for expression of up to four proteins were further described by Radcliffe and Mitrophanous [17]. Felipe et al. showed that 2A peptides worked in the yeast Saccharomyces cerevisiae. [18], Hecht et al [19] described poycistronic gene expression in yeast.